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Adenine Nucleoside Diphosphates Block Adaptation Of Proc. Natl. Acad. Sci. USA Vol. 90, pp. 2710-2714, April 1993 Neurobiology Adenine nucleoside diphosphates block adaptation of mechanoelectrical transduction in hair cells (auditory system/ear/molecular motor/myosin/vestibular system) PETER G. GILLESPIE AND A. J. HUDSPETH Center for Basic Neuroscience Research, University of Texas Southwestern Medical Center, Dallas, TX 75235-9039 Contributed by A. J. Hudspeth, December 16, 1992 ABSTRACT By adapting to sustained stimuli, hair cells in the internal ear retain their sensitivity to minute transient ATP ATP displacements. Because one model for adaptation asserts that this process is mediated by a myosin isozyme, we reasoned that we should be able to arrest adaptation by interfering with ATPase cycle though introduction of ADP into hair myosin's ADPj I, Weak- cells. During tight-seal, whole-cell recordings of transduction binding currents in cells isolated from bullfrog (Rana catesbeiana) states sacculus, dialysis with 5-25 mM ADP gave variable results. In half of the cells examined, the rate of adaptation remained unchanged or even increased; adaptation was blocked in the ADP remaining cells. Because we suspected that the variable effect of ADP resulted from the conversion of ADP to ATP by PTrl adenylate kinase, we employed the ADP analog adenosine 5'-[(3-thio]diphosphate (ADP[.JS]), which is not a substrate for adenylate kinase. Adaptation consistently disappeared in the Power stroke presence of 1-10 mM ADP[(ISJ; in addition, the transduction channels' open probability at rest grew from -0.1 to 0.8 or FIG. 1. Schematic depiction of myosin's ATPase cycle. In the more. Both effects could be reversed by 2 mM ATP. When used nucleotide-free or rigor state, the myosin molecule is tightly bound to an actin filament. ATP binds to myosin and promotes its rapid in conjunction with the adenylate kinase inhibitorP1,P5-bis(5'- dissociation from the filament; during the ensuing weak interaction adenosyl) pentaphosphate (Ap5A), ADP had effects similar to with actin, myosin cleaves ATP to ADPPi. As Pi is released, the those ofADP[(8S]. These results suggest that adaptation by hair myosin molecule rebinds tightly to the actin filament and performs a cells involves adenine nucleotides, and they lend support to the power stroke. Finally, ADP dissociates and the rigor state recurs. hypothesis that the adaptation process is powered by a myosin Because myosin and actin interact with high affinity, the rigor and motor. ADP-bound states are both capable of sustaining mechanical force. By responding to sounds and accelerations, the internal ear ity is regulated by the cytoplasmic Ca2+ concentration (12, performs one type of mechanoelectrical transduction. Hair 16, 18; reviewed in ref. 2). During adaptation to positive cells, the receptors of the cochlea and vestibular labyrinth, stimuli, it is supposed that Ca2+ enters a stereocilium through transform mechanical stimuli into electrical signals (reviewed open transduction channels and causes the motor to slip in refs. 1 and 2). The electrical response of each such cell is down a microfilamentous track, relieving tension in the gating produced by deflection ofa mechanically sensitive organelle, spring. During negative stimuli, when transduction channels the hair bundle, which emerges from the hair cell's apical are closed and the cytoplasmic Ca2+ concentration falls, the surface. A hair bundle comprises 20-300 actin-stiffened motor climbs up the track to restore tension. Because it could stereocilia, clustered together like the staggered pipes of an move along the microfilaments that form the cytoskeletal organ, and a single true cilium, the kinocilium. When a hair core of a stereocilium (19), a member of the myosin family bundle is displaced, transduction channels, perhaps as few as might well serve as the adaptation motor (12, 18, 20). one per stereocilium (3-5), are directly pulled open by To test the hypothesis that myosin molecules are respon- tension in gating springs within the hair bundle (6). Because sible for adaptation in the hair cell's transduction process, we these channels occur at the bundle's top (7, 8), it is probable sought to arrest the motors upon their tracks. A hair cell with that each gating spring is the thin, filamentous tip link that its adaptation motors stalled would be incapable of adjusting extends from the end ofa stereocilium to the side ofits tallest tip-link tension and should therefore maintain a constant neighbor (9, 10). transduction current in response to a protracted bundle When a hair bundle is subjected to a protracted displace- our approach to the ATPase cycle of ment, the cell's response adapts (11-16): the bundle's range displacement. Tailoring of responsiveness migrates toward the position to which the skeletal-muscle myosin (Fig. 1; reviewed in refs. 21 and 22), bundle has been moved. Adaptation is associated with me- we sought to arrest a myosin molecule in either of two states chanical changes within the hair bundle that are thought to in the cycle: rigor, in which the myosin molecule has no reflect adjustment of the tension in gating springs (12, 17). bound nucleotide, or the ADP-bound state, which ensues One model for adaptation posits that the tension in each after release of inorganic phosphate ion (Pj). A myosin gating spring is maintained by motor molecules whose activ- molecule confined to either of these states would be tightly bound to the actin fiament, able neither to climb nor to slip. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: ADP[,8S], adenosine 5'-[f-thio]diphosphate; ApsA, in accordance with 18 U.S.C. §1734 solely to indicate this fact. P1,P5-bis(5'-adenosyl) pentaphosphate. 2710 Downloaded by guest on September 23, 2021 Neurobiology: Gillespie and Hudspeth Proc. Natl. Acad. Sci. USA 90 (1993) 2711 Our results, some of which have appeared in preliminary I 1 form (23), demonstrate that ADP or an ADP analog blocks adaptation, and thus implicate adenine nucleotides in adap- Imax 1 + e&z(X-Xo)/kT' tation. Furthermore, the characteristics of the inhibition of adaptation are entirely consistent with the suggestion that the Here p is the channels' open probability, I is the transduction process is myosin based. current in response to a hair-bundle displacement X, Ima is the maximal transduction current, z is the channels' sensi- tivity to displacement, and Xo is the displacement at which MATERIALS AND METHODS half the channels are open; k is the Boltzmann constant and Adenine Nucleotide Solutions. Adenine nucleotides were T is the thermodynamic temperature. We used the fitted data obtained from Boehringer Mannheim. The concentrations of to estimate the fraction ofchannels open with the hair bundle ATP, ADP, and adenosine 5'-[,-thio]diphosphate (ADP[/S3]) in its resting position. To demonstrate that changes in the were determined by their absorbances at 259 nm on the basis channels' open probability were not artifactually produced of a molar extinction coefficient of 1.54 x 106 M-1m-1 (24). by drift of the stimulus probe, recording electrode, or micro- We estimated the concentration of Pl,P5-bis(5'-adenosyl) scope stage, we occasionally detached the probe and re- pentaphosphate (Ap5A) by assuming twice that molar extinc- attached it after confirming that the bundle was in its resting tion coefficient. The purity ofthe nucleotides was established position. by high-performance liquid chromatography on an anion- Transduction currents were measured with an amplifier exchange column (Mono Q, Pharmacia/LKB) with a 15-ml (EPC-7, List Electronics, Darmstadt, Germany) operated gradient of 0-1 M ammonium bicarbonate. Although the without series-resistance compensation. Stimulation was purities of ATP and ADP exceeded 95%, ADP[f3S] was controlled and responses were recorded by means of an contaminated by AMP (15%) and a small amount of ADP experimental interface (Indec Systems, Sunnyvale, CA) (0.1%). whose operation was programmed in BASIC-23 on a computer Electrophysiological Recording. Hair cells were isolated (PDP-11/73, Digital Equipment). The experimental environ- from bullfrog (Rana catesbeiana) sacculus after exposure of ment has been described in detail (8). the organ in situ to low-Ca2+ saline solution (10, 16). In a modification of the published procedure, 1 mM EGTA and 1 mM MgCl2 replaced 1 mM EDTA in the solution dripped into RESULTS the perilymphatic cistern. In addition, DNase treatment was We used whole-cell, tight-seal electrodes to record transduc- omitted and cells were mechanically dissociated in saline tion currents from hair cells isolated from the bullfrog's solution containing 100 iM CaC12. sacculus. When a pipette contained no nucleotide or milli- After solitary hair cells were firmly adsorbed onto a molar concentrations ofATP, our results accorded with those concanavalin A-coated coverslip in an experimental cham- from previous investigations of hair cells from this and other ber, they were maintained at 21°C in an oxygenated saline organs (reviewed in ref. 1). With the hair bundle in its resting solution containing 110 mM Na+, 2 mM K+, 4 mM Ca2+, 118 position, the cell's mechanically sensitive channels bore an mM Cl-, 3 mM D-glucose, and 5 mM Hepes at pH 7.25. Cells inward transduction current that was -15% of the maximum were observed through a 63x objective lens and Nomarski that could be evoked; the channels' open probability was thus differential-interference-contrast optics on an inverted mi- -0.15 (3). Moving the hair bundle progressively in the croscope (Zeiss model IM-35) with a rotating stage. positive direction, towards its tall edge, opened additional Transduction currents were recorded with whole-cell tight- channels until it evoked a transduction current that could be seal pipettes (25), whose axial resistances were 3-4 MfQ prior as great as -375 pA.
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